How can I improve the fixatives(glutraldehyde, paraformaldehyde) to obtain the ultrastructures of amoeba infected with M.ulcerans by TEM?
Srinivasan, what are the results you have gotten so far, can you provide more detail about the problem you are having? 4% glutaraldehyde in a cacodylate buffer should work, but be careful of the osmolarity and pH of your fixative cocktail. Are the cells freshwater or marine? A literature search for amebae ultrastructure should provide some recipes.
Sir, we are using freshwater amoeba with M.ulcerans.we would like to change different concentration of fixative mixtures (4% glutaraldehyde with 1%paraformaldehyde) to observe their morphological changes .
This study can help to understand the host and parasite relationship in Buruli ulcer in endemic areas.
During our primary studies we used 3% glutaraldehyde and 1%paraformaldehyde as fixative for acanthamoeba polyphagia cell with M ulcerans . we got high level of artifacts in the micrographs.
can you specify any literature related to this work.
Thank you
try this:
Interactions between Naegleria fowleri and Legionella pneumophila. A L Newsome, R L Baker, R D Miller and R R Arnold Infect. Immun. 1985, 50(2):449.
An easy googling (google scholar) immediately gives results:
http://www.nature.com/ismej/journal/v8/n8/full/ismej20145a.html
http://aem.asm.org/content/77/14/4974.full.pdf+html
Your protocol looks about right, but I should mention that freshness and purity of glutaraldehyde is of great importance. For best results you should use glut from EM suppliers.
I agree, freshness and purity of GA and PFA are very important. Have you fixed your samples with GA only? We use it (2.5 %) with very good results. For which time period do you fix your sample?
please how can i measure the quality of micrograph(specificity or sensitivity)?
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